We have continued our studies on polyamine biosynthesis and function in the eukaryote S. cerevisiae, emphasizing (1) the regulation of ornithine decarboxylase, a key enzyme in polyamine biosynthesis, and (2) the effect of polyamines on double-stranded RNA plasmids of yeast. (1) We have previously shown that a derepressed mutant strain of S. cerevisiae has a high ornithine decarboxylase activity when grown in the absence of amines, and no activity when grown in the presence of amines; however, immunoprecipitation of labeled extracts yields the same amount of enzyme protein. Thus post-translational modification of the enzyme is an important control in yeast. We are studying the specific modification which occurs. Cultures labeled with C-14 or H-3 spermidine and spermine showed no incorporation of labeled amines into the ornithine decarboxylase protein. This proves that transglutaminase activity, which other investigators have found to incorporate polyamines into the mammalian enzyme in vitro, with a loss of activity, does not play a role in the amine-dependent inactivation of the yeast enzyme. We have recently found that the yeast enzyme is a phosphoprotein. Cells grown in the presence or absence of polyamines have the same P-32 incorporation. Thus, we have not found any evidence for phosphorylation or dephosphorylation in the regulation of ornithine decarboxylase activity of yeast. We are continuing with attempts to determine the controlling modification. (2) We have investigated the requirement for polyamines in the maintenance of double-stranded RNA plasmids. Two killer plasmids (M1 and M2) are not maintained when yeast cells carrying these plasmids are made completely deficient in spermidine and spermine. Putrescine alone cannot maintain these plasmids. One double-stranded RNA plasmid (L-A-E) requires amines also, but putrescine alone is sufficient to maintain this plasmid. No amine requirement has been found for some other yeast RNA plasmids (L-A-HN), as well as Rho and Psi. Thus the maintenance of the yeast killer RNA plasmids M1 and M2 is absolutely dependent on spermidine or spermine, but cannot be satisfied by putrescine; the maintenance of RNA plasmid L-A-E also requires amines, but putrescine can satisfy this requirement, as well as spermidine or spermine.